Throughout this application various publications are referred to in parenthesis. Citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference in their entireties into the subject application to more fully describe the art to which the subject application pertains.
During an idealized 28-day human menstrual cycle, the endometrium undergoes well-timed changes in preparation for embryo implantation. The follicular or proliferative phase is separated by ovulation on day 14 from the luteal or secretory phase. The endometrium is only receptive for a short two-day period during luteal days 20-24 (Wilcox et al., 1999). Inaccurate identification of this implantation window is a major cause for the low success rate in artificial reproductive technologies (Norwitz et al., 2001).
These temporal changes of the endometrium are evident on the tissue and epithelial cell level. In fact, histological changes have been the gold standard for endometrial dating for the past 50 years but their value has recently been questioned (Coutifaris et al., 2004; Murray et al., 2004; Noyes et al., 1950). Among the ultrastructural hallmarks of endometrial epithelial cells are giant mitochondria, subnuclear glycogen deposits, pinopodes, and nucleolar channel systems (NCSs) (Martel, 1981; Spornitz, 1992). Whereas giant mitochondria and subnuclear glycogen deposits appear in the early luteal phase, pinopodes and NCSs more closely overlap with the mid luteal window of implantation and could serve as potential markers (Clyman, 1963; Nikas et al, 1995).
NCSs were discovered in the nuclei of endometrial epithelial cells using transmission electron microscopy, which is still their only method of identification (Dubrauszky and Pohlmann, 1960). NCSs are small globular structures of about 1 μm in diameter and consist of three components, intertwined membrane tubules embedded in an electron dense matrix, and an amorphous core that is separated from the nucleoplasm by the tubules and matrix (Clyman, 1963; Moricard and Moricard, 1964; Terzakis, 1965). Using histochemical labeling, the activity of glucose-6-phosphatase, a marker enzyme of endoplasmic reticulum, was documented in the lumen of the membrane tubules indicating their derivation from this cytoplasmic organelle, apparently through the contiguous nuclear envelope (Kittur et al., 2007).
Understanding of nuclear structure and function has advanced significantly (Stewart et al., 2007; Terry et al., 2007; Trinkle-Mulcahy and Lamond, 2007). Nuclear pore complexes (NPCs) perforate the nuclear envelope at the sites where the outer and inner nuclear membranes fuse and are thought to serve as the sole portal between nucleus and cytoplasm. The NPCs are large complex protein assemblies consisting of 35 or so proteins (nucleoporins) present in multiple copies and arranged in partial symmetry across the envelope and around the pore. Although some nucleoporins can exchange off NPCs during interphase and some concentrate in kinetochores during mitosis when NPCs disassemble, they are generally restricted to intact NPCs (Belgareh et al., 2001; Rabut et al., 2004). Whereas the outer membrane and the perinuclear space mirror the proteins of the attached endoplasmic reticulum, the protein composition of the inner nuclear membrane is distinct. Inner membrane proteins anchor the lamina (an intermediate filament meshwork lining the nucleoplasmic side) and/or chromatin at the nuclear envelope. Several of these proteins, including lamins (proteins of the lamina), are mutated in inherited diseases ranging from muscular dystrophies to progeria (premature aging) (Stewart et al., 2007).
Several lines of evidence suggest a role for NCSs in the preparation of the endometrium for reception of the embryo. NCSs have strictly been observed post ovulation, only on cycle days 16-24, and are not detected in pregnancy (Clyman, 1963). They appear to be induced by progesterone and are sensitive to oral and intrauterine contraceptives (Azadian-Boulanger et al., 1976; Feria-Velasco et al., 1972; Kohorn et al., 1970; Kohorn et al., 1972; Pryse-Davies et al., 1979; Roberts et al., 1975; Wynn, 1967). Finally, in several cases of unexplained infertility the absence or delayed appearance of NCSs was noted as the sole abnormal endometrial parameter (Dockery et al., 1996; Gore and Gordon, 1974; Kohorn et al., 1972). Despite this and additional evidence, NCSs have been neglected as potential markers or prerequisites for implantation. This can be mostly attributed to difficulty of their detection requiring transmission electron microscopy, which is further complicated by their small size and the perception that only about 5% of all endometrial epithelial cells develop NCSs (Novotny et al., 1999; Ryder et al., 1995). Accordingly, a method is needed that can be readily used to mark the window of uterine receptivity.